الفهرس 
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Abstract
The oxalotrophic bacteria is of great importance in reducing
urinary excretion of oxalic acid, since the high oxalate level is one of the important virulence factors for hyperoxaluria, urolithiasis, cardiomyopathy, and renal failure. Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to carbon dioxide and formate. The oxalate decarboxylase has commercial uses such as in the brewing industry or for agronomic uses such as to reduce susceptibility of a plant to oxalic acid and phytopathogens, clinical oxalate determination, bioremediators, plant growth promoters, antinematodal and antifungal activity. In the current work, mesophilic bacteria were isolated from different fertile soil samples obtained from several governorates. Isolation resulted in several isolates. Screening of the bacterial isolates was carried out according to their ability to degrade oxalate.
Screening resulted in 16 isolates designated as: SK- 1,SK- 2, SK-
3,SOG- 1, SOG- 2, SOG- 3, TG- 1, TG- 2, TG- 3, PG- 1, PG- 2,
MG- 1, MG- 2, CG- 1, CG- 2, CG- 3 and SOG- 1. Among the
selected isolate 6 isolate showed the highest potential of oxalate
degradation. Characterization of the selected isolate showed that it was highly sensitive to Ofloxacin (5μg), Ceftazidime (30 μg), Claforan cefotaxime (30 μg), Cefoperazone (75 μg), Ciprofloxacin (5 μg), moderately sensitive to Tobramycin (10 μg), Sulphamethxazole trimethoprime (25 μg), Cefepime (30 μg), Erythromycin(15 μg), clavulanic acid(30 μg) and resistant to Ampicillin sulbactam (20 μg) and Cefexime (5 μg). So-G1 isolate was found to be highly sensitive to mercury, the descending order of metal toxicity observed was Hg >co >cu > Cr2O7> Ni > Zn >Pb. The selected isolate was subjected to phenotypic identification using API 20 NE system, genotypic identification and fingerprinting using randomly amplified polymorphic DNA (RAPD). The results of commercial API 20 NE showed excellent identification of Burkholderia cepacia, the percentage of identity 99.8% for 5167777 profile. The polymorphisms amplified by the nine RAPD primers from DNA extracted from B. cepacia showed a DNA fingerprint ranging from 0 to 9 bands, over a size range of 344 bp to 3280 bp. Optimization of oxalate decarboxylase production revealed that the optimal environmental conditions were: 28- 37 C°, pH 9 with shaking (105rpm) at the 3rd day of incubation and the optimal nutritional conditions were: 4g/l potassium oxalate, inositol and sorbitol are the best carbon sources, ammonium nitrate and casein are the best nitrogen sources and zinc is highly promoted the growth and
oxalate degrading activity but mercury has inhibitory effect where thedecreasing order of heavy metal effect observed was Zn > Co> Ni >Cr= Cu > Pb.
The selected isolate exhibited good influence in decreasing
the urinary oxalate level and also exhibited good influence in
degrading the oxalate stone for both male and female calcium
oxalate stone formers patients in Ain Sham specialized hospital withdifferent ages.
Time course for the enzyme substrate reaction revealed that
the optimal reaction time required to complete oxalate / oxalate
decarboxylase reaction leading to oxalate consumption is 15 minutes.
Purification of intracellular oxalate decarboxylase was
conducted as follow:
· Fractional precipitation of oxalate decarboxylase by
ammonium sulphate at 40% saturation ammonium sulphate.
· Dialysis of precipitated oxalate decarboxylase in order to
eliminate undesired materials.
· Oxalate decarboxylase was further purified using gel filtration
chromatography.
· Data revealed that the bacterial oxalate decarboxylase enzyme
was homogeneous showing pure single band with molecular weight 30 kDa according to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis with Coomassie staining.
· Characterization of oxalate decarboxylase revealed that the
optimal value at pH 6, Zn and Mn are the best metal ions for oxalate decarboxylase activity whereas Hg is the most potent inhibitor for the enzyme.
· Amino acid composition of oxalate decarboxylase was done.
Results of amino acid analysis showed that the most abundant amino acid is Alanine and the less frequent amino acid is Asparagine . Also it was found that the number of basic amino acids was 47 and number of acidic amino acids was 54. The polar amino acids are 186 and the non-polar 232 amino acid.
· The isoelectric point is 6.27.
· Checking of any plasmid present in Burkholderia cepacia
showed that there were no extra plasmid in the bacteria but only
megaplasmid (genomic) DNA with a size of 12,437.6 bp.
· Comparison of genomic and plasmid DNA showed that both
have the same size indicating that only genomic DNA present in the bacteria without any extra plasmids.
· Oxalate decarboxylase gene was amplified from genomic
DNA using PCR. In PCR procedures, forward and reverse primers, which contain the restriction sites, were used. After electrophoresis, expected size (1305 bp) was obtained.
· Purified DNA from gel was subjected to DNA sequence
analysis. Results obtained from DNA sequence analysis were further analyzed through sequence similarity search as presented using BLAST option of internet
(http://www.ncbi.nlm.gov/BLAST). Using the blast option of the
Internet, a search for similarity of the oxalate decarboxylase gene in the Genebank and other associated sites was done . Search results showed that the obtained sequence can detect oxalate decarboxylase gene from many B. cepacia genomes.
· The translated amino acid sequence analysis was done.